pH值和盐浓度对金属螯合亲和层析分离人胰高血糖素样肽-1融合蛋白的影响

目的探讨pH值和盐浓度对金属螯合亲和层析(Immobilized metal ion affinity chromatography,IMAC)分离含His-Tag标签的人胰高血糖素样肽-1(Glucagon-like peptide-1,GLP-1)融合蛋白的影响,并确定最佳洗脱条件。方法在0.50mol/L盐浓度条件下,分别取pH6.0、7.0、7.8和9.04种缓冲液进行咪唑梯度洗脱;在合适的pH值条件下,分别取氯化钠浓度0.25、0.50和0.75mol/L3种缓冲液进行咪唑梯度洗脱。分析在不同pH值及盐浓度条件下洗脱融合蛋白所需要的咪唑浓度及回收率。结果在所考察的pH值范围内,洗脱融合蛋白所需的咪唑浓度随洗脱液pH值的升高而逐渐降低,当洗脱液pH值为7.8时,融合蛋白的分离效果最佳,大部分杂蛋白被0～40mmol/L咪唑洗脱液去除且不含融合蛋白,而绝大部分融合蛋白在咪唑浓度达80～200mmol/L时被洗出,总回收率达(83.7±1.0)%,且比例较高;在此pH值条件下,氯化钠浓度达0.75mol/L,才会影响融合蛋白的回收率,氯化钠浓度为0.25或0.50mol/L的洗脱液洗出融合蛋白的效果相近;在pH7.8,含0.25mol/L氯化钠的洗脱体系下,用50mmol/L咪唑溶液洗脱杂蛋白,200mmol/L咪唑溶液洗脱融合蛋白,融合蛋白的回收率和比例分别可达(85.2±2.0)%和(80.5±1.0)%。结论在所考察的pH值和盐浓度范围内,提高洗脱液pH值和盐浓度均可降低洗脱融合蛋白需要的咪唑量,但也会降低融合蛋白的回收率。

Effects of pH Value and Salt Concentration on Purification of Human Glucagonlike Peptide-1 Fusion Protein by Immobilized Metal Ion Affinity Chromatography

Objective To investigate the effects of pH value and salt concentration on purification of human glucagon-like peptide-1(GLP-1)fusion protein with His-Tag by immobilized metal ion affinity chromatography(IMAC)and optimize the condition for elution.Methods The fusion protein on IMAC column was eluted by imidazole gradient elution using buffers containing 0.50 mol/L sodium chloride at pH 6.0,7.0,7.8 and 9.0 respectively,and the imidazole concentration required for elution as well as the recovery rate of fusion protein were analyzed.Then,at the optimal pH value,the fusion protein was eluted with the buffers containing 0.25,0.50 and 0.75 mol/L sodium chloride respectively and analyzed for the imidazole concentration required and the recovery rate.Results Within the range of pH values evaluated,the imidazole concentration required for elution of fusion protein decreased with the increasing pH value.At pH 7.8,most of foreign protein free of fusion protein was removed by 0 ~ 40 mmol/L imidazole eluent,while most of fusion protein was eluted by 80 ~ 200 mmol/L imidazole eluent,which reached a total recovery rate of(83.7 ± 1.0)% and a high percentage in total protein,indicating that 7.8 was the optimal pH value.However,at the optimal pH value,only the sodium chloride concentration of 0.75 mol/L showed effect on the recovery rate of fusion protein,while the elution efficacies of fusion protein at sodium chloride concentrations of 0.25 and 0.50 mol/L were similar.Hereby,an elution system was developed,in which foreign protein was eluted with 50 mmol/L while fusion protein with 200 mmol/L imidazole eluent at pH 7.8 and a sodium chloride concentration of 0.25 mol/L.By using the system,the recovery rate and percentage in total protein of fusion protein reached(85.2 ± 2.0)% and(80.5 ± 1.0)% respectively.Conclusion Within the range of pH value and salt concentration evaluated,both increased pH value and salt concentration of eluent decreased the imidazole concentration required,while decreased the recovery rate of fusion protein.